The present invention relates to multiplexed chromatographic methods. Such methods can be used for detecting mutations within a population of nucleic acid samples. They can also be used for genotyping and haplotyping.
Deciphering the genetic code and the establishment of the structure of deoxyribonucleic acid (DNA) in the early 1960s initiated a revolution in modem biology. Since that time, numerous methods have been developed for the isolation, analysis, and manipulation of nucleic acid samples.
One such method developed for the analysis of nucleic acid samples is polymerase chain reaction amplification (referred to herein as xe2x80x9cPCRxe2x80x9d). PCR is an in vitro method for replicating a defined (or target) DNA molecule to increase the amount of total DNA for subsequent analysis, such as sequencing, Northern and Southern hybridizations, and the like. Typically, the amount of total DNA increases exponentially, i.e., it is amplified. Thus, PCR can be utilized in connection with a variety of techniques when it is desirable to manipulate and analyze genetic information of a DNA molecule that may be in low copy numbers. For example, PCR may be used in connection with cloning genes, sequencing, genome mapping, site directed mutagenesis, diagnostic assays, environmental monitoring, to name a few.
Due to the vast amount of genetic information that is capable of being generated and gathered, intense efforts are underway to develop new and faster methods of DNA detection, sizing, quantification, sequencing, and gene identification including the mapping of human disease genes. Although the efficiency of these processes has been improved by automation, more efficient and less expensive methods must still be developed to efficiently carry out genomic-scale DNA analyses.
The detection of polymorphisms is becoming increasingly important, particularly in gene mapping. Although the majority of DNA in higher organisms is identical in sequence among the chromosomes of different individuals, a small fraction of DNA is variable or polymorphic in sequence. It is this variation which is the essence of genetic science and human diversity. Mutations arise either due to environmental effects or randomly during replication as a change in the sequence of a gene, with different mutations having differing consequences. In fact, single base pair changes, called single nucleotide polymorphisms (SNPs) are frequent in the human genome. The level of genetic variation between two individual sequences is estimated to be on average one difference per 1,000 base pairs. Based on this estimate, the average amount of genomic variation between individuals is about 3 million base pairs. It is this normal polymorphism, which provides the basis for some of the emerging gene localization strategies.
As the sequences of greater numbers of genes are identified, the detection of specific polymorphisms in such genes and the correlation to specific diseases can provide an invaluable tool in the screening and detection of diseases. Diagnostic screening methods for polymorphisms are also useful in the detection of inherited diseases in which either a single point mutation or a few known mutations account for all cases (e.g., sickle cell disease). Presently, over 200 genetic disorders can be diagnosed using recombinant DNA techniques. Such techniques have also been used for other purposes, such as for forensic screening.
Presently used methods for screening for polymorphic sites within a gene include single-stranded conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), RNase A cleavage, chemical cleavage, allele specific oligonucleotides (ASOs), ligase mediated detection of mutations, and denaturing high performance liquid chromatography.
Briefly, in single-stranded conformation polymorphism (SSCP), DNA is denatured and then immediately run on a non-denaturing gel. The secondary structures of wild-type strands or mutant single strands differing by a single base are usually sufficiently different to result in different migration rates on polyacrylamide gels.
In denaturing gradient gel electrophoresis (DGGE), either homoduplex or heteroduplex double stranded DNA is electrophoresed under denaturing conditions of increasing concentration until the last domain is denatured, and migration of the DNA through the gel is retarded. DNA sequences differing by a single base pair migrate at different rates along the gel, thereby allowing detection of a polymorphic site, if present.
RNase A cleavage utilizes the enzyme ribonuclease A to cut RNA-DNA hybrids wherever there is a mismatch between a nucleotide in the RNA strand and the corresponding nucleotide in the DNA strand. The chemical cleavage method is based upon a similar principle but uses hydroxylamine and osmium tetroxide to distinguish between mismatched C or T nucleotides, respectively. The position of the mismatch (e.g., the mutation) is defined by sizing on gel electrophoresis after cleavage at the reactive position by piperidine.
Allele-specific oligonucleotide probes (ASOs) are probes that are designed to hybridize selectively to either a normal or a mutant allele, where the probes are developed to distinguish between the normal and mutant sequence. This is done by altering the stringency of hybridization to a level at which each of the oligonucleotides will anneal stably only to the sequence to which it is perfectly complementary and not to the sequence with which it has the single mismatch.
The ligase-mediated method for detecting mutations makes use of the fact that the ends of two single strands of DNA must be exactly aligned for DNA ligase to join them. In utilizing this technique, oligonucleotides complementary to the target sequence, 5xe2x80x2 to and including the mutation site, are synthesized and labeled. A third oligonucleotide complementary to the common sequence 3xe2x80x2 to the mutation site is synthesized and also labeled. The oligonucleotides are then hybridized to strands of the target. In cases in which the 5xe2x80x2 and 3xe2x80x2 oligonucleotides form a flush junction that can be joined by DNA ligase, ligation occurs. However, a single base pair mismatch occurring between the normal 5xe2x80x2 oligonucleotide and the mutation site is sufficient to prevent the ligase from acting and can readily be detected.
A common approach to analysis of DNA polymorphisms relies on variations in the lengths of DNA fragments produced by restriction enzyme digestion. The polymorphisms identified using this approach are typically referred to as restriction fragment length polymorphisms or RFLPs. Polymorphisms involving variable numbers of tandemly repeated DNA sequences between restriction enzyme sites, typically referred to as microsatellites or variable numbers of tandem repeats (VNTRs), have also been identified.
While existing methods may locate polymorphic sites, point mutations, insertions and deletions on a gene, many of these methods are generally time consuming, necessitate multiple handling steps, require product purification, are not readily adaptable to automation, have limitations in sensitivity and accuracy, and are typically limited to detection in small-sized nucleic acid fragments.
Furthermore, existing methods typically do not yield haplotype information (i.e., linked polymorphism) without the use of multiple, and often complicated, steps that may incorporate toxic chemicals. See, for example, Verpy et al., Proc. Natl. Acad. Sci., USA, 95, 1873-1877 (1994).
Denaturing high performance liquid chromatography for separating heteroduplex (double-stranded nucleic acid molecules having less than 100% sequence complementarity) and homoduplex (double-stranded nucleic acid molecules having 100% sequence complementarity) nucleic acid samples (e.g., DNA or RNA) in a mixture is described in U.S. Pat. No. 5,795,976 (Oefler et al.). In the separation method, a mixture containing both heteroduplex and homoduplex nucleic acid samples is applied to a stationary reversed phase support. The sample mixture is then eluted with a mobile phase containing an ion-pairing reagent and an organic solvent. Sample elution is carried out under conditions effective to at least partially denature the duplexes and results in the separation of the heteroduplex and homoduplex molecules. Also disclosed is the amplification of homoduplex and heteroduplex molecules using the polymerase chain reaction. The amplified DNA molecules are denatured and renatured to form a mixture of heteroduplex and homoduplex molecules prior to separating the molecules. This method can be used to run multiple samples at once as long as the different samples do not co-elute in time, which is referred to as multiplexing in time. The disadvantage of this is that each analytical run takes longer than a single non-multiplexed run.
What is yet needed is a relatively rapid method for identifying nucleic acids, specifically for distinguishing individual polymorphic nucleic acid molecules. What is also needed is a relatively rapid method for genotyping and haplotyping that involves relatively fewer steps, is capable of automation, and generates information relatively quickly. The present invention provides such methods. In a preferred embodiment, a method of the present invention can be used to distinguish individual PCR amplicons (also referred to as PCR products herein) from a PCR reaction mixture. Significantly and advantageously, the present invention involves the use of multiplexed denaturing liquid chromatography, particularly multiplexed denaturing high performance liquid chromatography.
As used herein, xe2x80x9cmultiplexingxe2x80x9d or xe2x80x9cmultiplexedxe2x80x9d refers to the ability to run multiple (different) samples substantially simultaneously under similar conditions and be able to reconstruct the data individually for each sample. It involves using a detectable label or tag that can be monitored spectrally. In essence, the method of the present invention involves spectral multiplexing. This is distinct from multiplexing in time because in the present invention all samples can be run in the same time period in which one sample could be run (i.e., substantially simultaneously). In a preferred embodiment of the invention, using fluorescence multiplexing, the samples are exposed to radiation having a wide range of wavelengths, individual wavelengths are monitored, and then the mixed signals, which are spectrally resolved, are reconstructed according to their spectral properties.
In one embodiment of the present invention, a method is provided for separating nucleic acid (e.g., DNA and RNA) samples in a test mixture. The method includes: providing a test mixture of a plurality of nucleic acid samples, wherein each sample is labeled with a spectrally detectable tag; applying the test mixture to a reversed phase support; eluting the mixture under partially denaturing conditions to separate at least one nucleic acid sample from the test mixture (preferably, all of the nucleic acid samples are separated from each other during the elution); and detecting spectrally resolved signals produced by the nucleic acid samples labeled with the detectable tags. Preferably, the nucleic acid samples include PCR products (i.e., PCR amplicons), particularly heteroduplexes and homoduplexes. Preferably, the reversed phase solid support is in a high performance liquid chromatography (HPLC) column.
Preferably, the tag is selected from the group of electromagnetic and electrochemical tags, and more preferably, the tag is selected from the group of spectrophotometric and spectrofluorometric tags, and most preferably, the tag is a spectrofluorometric tag. In a particularly preferred embodiment, the detectable label or tag is a fluorescent dye. Preferably, the test mixture is formed by combining a different fluorescent dye with each nucleic acid sample to form a labeled nucleic acid sample; and combining the labeled nucleic acid samples to form the test mixture. Preferably, a different fluorescent dye is added to each nucleic acid sample separately during polymerase chain reaction amplification of the nucleic acid sample prior to combining the nucleic acid samples to form the mixture.
In the present method, the eluting step includes the use of a mobile phase containing an ion-pairing agent and optionally an organic solvent. Examples of ion-pairing agents include amines such as lower allcyl primary, secondary, and tertiary amines, ammonium salts such as lower trialkylammonium salts (e.g., triethylammonium acetate) and lower alkyl quaternary ammonium salts.
A variety of methods can be used for partial denaturation of the mixture of nucleic acid samples (e.g., PCR amplicons). For example, temperatures of about 50xc2x0 C. to about 80xc2x0 C. can be used. Alternatively, a chemical reagent for denaturation can be used in the mobile phase.
Preferably, detecting spectrally resolved signals involves the use of spectrophotofluorometric methods of detection in which excitation and emission wavelengths can be independently chosen. In such methods, emission wavelengths can be detected at very low concentrations, often at less than about 1 nanomolar concentrations. Thus, in preferred embodiments, the methods of the present invention include detecting spectrally resolved signals using a spectrophotofluorometric HPLC detector.
Typically, detecting spectrally resolved signals produced by the nucleic acid samples labeled with the detectable tags includes passing the separately labeled nucleic acid samples through a detection zone of a detector substantially simultaneously, each sample generating a specific signal which is spectrally resolved from the specific signals of the other nucleic acid samples. Preferably, the detector excites the detectable tags at one wavelength and detects emissions at multiple wavelengths. Alternatively, the detector can excite the detectable tags using zero-order excitation.
In one preferred embodiment, the present invention provides a method for detecting genotypic variations. The method includes: providing a pre-mixture that includes one unlabeled nucleic acid sample and two or more reference genotypes of labeled nucleic acid, wherein each labeled nucleic acid is labeled with a different spectrally detectable tag and the unlabeled nucleic acid is present in an excess amount relative to the total amount of labeled nucleic acid; denaturing and reannealing the pre-mixture to form a test mixture comprising labeled/unlabeled nucleic acid duplexes; applying the test mixture to a reversed phase support; eluting the test mixture under partially denaturing conditions to separate at least one of the labeled/unlabeled nucleic acid duplexes from the test mixture; detecting spectrally resolved signals produced by the labeled nucleic acid to define an elution profile for each tag; and deducing from the elution profiles the composition of the unlabeled nucleic acid sample. Preferably, the tag is a spectrally detectable tag selected from the group of an electromagnetic tag and an electrochemical tag.
In another preferred embodiment, the present invention provides a method for determining haplotypes. The method includes: providing a pre-mixture that includes one unlabeled nucleic acid sample and four or more reference haplotypes of labeled nucleic acid, wherein each labeled nucleic acid sample includes two or more alleles, each reference haplotype is labeled with a different detectable tag, and the unlabeled nucleic acid is present in an excess amount relative to the total amount of labeled nucleic acid; denaturing and reannealing the pre-mixture to form a test mixture comprising labeled/unlabeled nucleic acid duplexes; applying the test mixture to a reversed phase support; eluting the test mixture under partially denaturing conditions to separate at least one of the labeled/unlabeled nucleic acid duplexes from the test mixture; detecting spectrally resolved signals produced by the labeled nucleic acid to define an elution profile for each tag; and deducing from the elution profiles the composition of the unlabeled nucleic acid sample. Preferably, the tag is a spectrally detectable tag selected from the group of an electromagnetic tag and an electrochemical tag.
The following terms, as used herein, have the meanings as indicated:
xe2x80x9cReversed phase supportxe2x80x9d refers to a stationary support (including the base material and any chemically bonded phase) for use in liquid chromatography, particularly high performance liquid chromatography (HPLC), which is less polar (e.g., more hydrophobic) than the starting mobile phase.
xe2x80x9cIon-pair (IP) chromatographyxe2x80x9d refers to a chromatographic method for separating samples in which some or all of the sample components contain functional groups which are ionized or are ionizable. Ion-pair chromatography is typically carried out with a reversed phase column in the presence of an ion-pairing reagent.
xe2x80x9cIon-pairing reagentxe2x80x9d is a reagent which interacts with ionized or ionizable groups in a sample to improve resolution in a chromatographic separation. An xe2x80x9cion-pairing agentxe2x80x9d refers to both the reagent and aqueous solutions thereof An ion-pairing agent is typically added to the mobile phase in reversed phase liquid chromatography for optimal separation. The concentration and hydrophobicity of an ion-pairing agent of choice will depend upon the number and types (e.g., cationic or anionic) of charged sites in the sample to be separated.
xe2x80x9cHomoduplex moleculesxe2x80x9d are typically composed of two complementary nucleic acid strands.
xe2x80x9cHeteroduplex moleculesxe2x80x9d are typically composed of two complementary nucleic acid strands (e.g., DNA or RNA), where the strands have less than 100% sequence complementarity. Functionally, in a mixed population of homoduplex and heteroduplex molecules, shorter strands (e.g., typically about less than 50 base pairs in size) of heteroduplex molecules elute as peaks corresponding to their respective denatured single strands under select denaturing conditions using reversed phase ion-pairing chromatography, separable from those of homoduplex molecules. In a mixed population of homoduplex and heteroduplex molecules larger than about 50 base pairs in length, heteroduplex molecules typically elute with shorter retention times than those of homoduplexes of essentially the same size under select denaturing conditions using reversed phase ion-pairing chromatography.
A heteroduplex molecule that is xe2x80x9cpartially denaturedxe2x80x9d under a given set of chromatographic conditions refers to a molecule in which several complementary base pairs of the duplex are not hydrogen-bond paired, such denaturing typically extending beyond the site of the base-pair mismatch contained in the heteroduplex, thereby enabling the heteroduplex to be distinguishable from a homoduplex molecule of essentially the same size. In accordance with the present invention, such denaturing conditions may be either chemically (e.g., resulting from pH conditions) or temperature-induced, or may be the result of both chemical and temperature factors.
xe2x80x9cGenotypexe2x80x9d refers to the genetic constitution of a cell or an organism such that expression of the genetic constitution gives rise to an organism""s physical appearance. In general, the genetic constitution means a gene, wherein alternative forms of the gene are called xe2x80x9calleles.xe2x80x9d Typically, an allele is carried at a genetic locus, or position, on an organism""s chromosome. xe2x80x9cGenotypingxe2x80x9d refers to detecting which alleles are present in a given individual.
xe2x80x9cHaplotypexe2x80x9d refers to a set of closely linked alleles on a specific chromosome carried by an individual and inherited as a unit, such as the alleles of the major histocompatibility complex on human chromosome number 6. xe2x80x9cHaplotypingxe2x80x9d refers to detecting a change or mutation that may be within one or all of the linked alleles (i.e., the haplotype).
xe2x80x9cPrimerxe2x80x9d refers to an oligonuleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a target nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization (such as a DNA polymerase) and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products (referred to herein as xe2x80x9cPCR productsxe2x80x9d and xe2x80x9cPCR ampliconsxe2x80x9d) in the presence of the polymerization agent. Primers are preferably selected to be xe2x80x9csubstantiallyxe2x80x9d complementary to a portion of the target nucleic acid sequence to be amplified. This typically means that the primer must be sufficiently complementary to hybridize with its respective portion of the target sequence. For example, a primer may include a non-complementary nucleotide portion at the 5xe2x80x2 end of the primer, with the remainder of the primer being complementary to a portion of the target sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with a portion of the target sequence to hybridize therewith, and thereby form a template for synthesis of the extension product.